Detection of nucleic acids has grown in recent years as a means for early detection of genomic features, infectious agents and various organisms which are present in very small quantities in a human, plant, microbial or animal test specimen. Detection procedures are normally based on the concept of complementarity whereby two DNA strands are bound together by hydrogen bonds and other forces between complementary nucleotides (which are known as nucleotide pairs).
A DNA molecule is normally quite stable, but the strands can be separated or denatured by certain conditions, such as heating. The denatured strands will reassociate only with another strand having a complementary sequence of nucleotides.
Much research has been carried out to find ways to detect only a few molecules of a DNA. Various procedures are known and have been used for more than a decade to amplify or greatly multiply the number of nucleic acids in a specimen for detection. Such amplification techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR) and others which are less developed.
Both chain reactions—in particular the polymerase chain reaction (PCR) process—for amplifying nucleic acid sequences are well known in the art and disclosed in U.S. Pat. Nos. 4,683,202; 4,683,195; and 4,965,188.
In each cycle of a PCR amplification, a double-stranded target sequence is denatured, primers are annealed to each strand of the denatured target, and the primers are extended by the action of a DNA polymerase. Specificity of amplification depends on the specificity of primer hybridisation, also referred to primer annealing. Primers are selected to be complementary to, or substantially complementary to, sequences occurring at the 3′ end of each strand of the target nucleic acid sequence. Under the elevated temperatures used in a typical PCR, the primers hybridize only to the intended target sequence. However, amplification reaction mixtures are typically assembled at room temperature, well below the temperature needed to ensure primer hybridization specificity. Under such less stringent conditions, the primers may bind non-specifically to other only partially complementary nucleic acid sequences (or even to other primers) and initiate the synthesis of undesired extension products, which can be amplified along with the target sequence. Amplification of the non-specific primer extension products can compete with amplification of the desired target sequences and can significantly decrease the efficiency of the amplification of the desired sequence. Problems caused by non-specific amplification are discussed further in Chou et al., 1992, Nucleic Acids Research 20(7):1717-1723, incorporated herein by reference.
Non-specific amplification can be reduced by reducing the formation of extension products from primers bound to non-complementary target sequences prior to the start of the reaction. In one method, referred to as a “hot-start” protocol, one or more critical reagents are withheld from the reaction mixture until the temperature is raised sufficiently to provide the necessary hybridization specificity. In this manner, the reaction mixture cannot support primer extension during the time that the reaction conditions do not ensure specific primer hybridization.
Hot-start methods can be carried out manually by opening the reaction tube after the initial high temperature incubation step and adding the missing reagents. However, manual hot-start methods are labor intensive and increase the risk of contamination of the reaction mixture. Hot-start methods which use a heat labile material, such as wax, to separate or sequester reaction components are described in U.S. Pat. No. 5,411,876, incorporated herein by reference, and Chou et al., 1992, supra. In these methods, a high temperature pre-reaction incubation melts the heat labile material, thereby allowing the reagents to mix.
Methods and reagents for amplifying nucleic acid using a primer-based amplification reaction which provide a simple and economical solution to the problem of non-specific amplification are known from the state of the art (e.g. U.S. Pat. Nos. 5,773,258 or 6,183,998). The methods use a reversibly inactivated thermostable enzyme which can be reactivated by incubation in the amplification reaction mixture at an elevated temperature. Non-specific amplification is greatly reduced because the reaction mixture does not support primer extension until the temperature of the reaction mixture has been elevated to a temperature which insures primer hybridization specificity.
Another problem does arise from the fact that human and animal specimens contain many different nucleic acids, some of which are endogenous (or natural) to the person or animal, and others which are produced because of some abnormal condition, such as from the presence of an infectious agent or an oncogenic condition. Such nucleic acids are usually present in very low concentrations compared to endogenous nucleic acids. They are sometimes referred to as “low copy number” nucleic acids. By comparison, the endogenous nucleic acids are usually present in high concentrations and may be referred to as “high copy number” nucleic acids. One such example is human β-globin DNA. Frequently, in using PCR, two or more nucleic acids present in the specimen are amplified at the same time in the same reaction container. This is identified herein as “co-amplification”. This process requires that primers for each nucleic acid to be amplified must be simultaneously present in the container.
When both low and high copy target nucleic acids are amplified in such situations, amplification of the low copy target nucleic acid is often inhibited. This is due to the saturation of the amplifying enzyme (such as DNA polymerase) by the high copy target nucleic acid during the later cycles of amplification. False negative results for the presence of the low copy target nucleic acid are likely, with possibly serious consequences.
A similar problem arises if such co-amplification reactions shall be carried out for quantitative measurements of nucleic acid molecules such as quantitative PCR. Quantitative PCR and quantitative reverse transcription-polymerase chain reaction (RT-PCR) is used for determining the amount of specific DNA or RNA molecules in biological samples. The method has evolved from a low-throughput gel-based analysis to the use of fluorescence techniques that do not require the separation of the reaction product on a gel (‘closed tube’ format). The amount of DNA or cDNA amplified by a PCR is then proportional to an increase in the fluorescent signal. The amount of starting DNA is then determined by analyzing the fluorescence at each cycle of PCR (real-time or online PCR), or after the PCR (end-point detection). These fluorescent techniques which most often comprise fluorescent labelled sequence-specific probes are faster and can be less expensive since they do not require post-PCR. These methods also reduce contamination of the laboratory with PCR amplicon molecules that may interfere with subsequent assays. Background on quantitative PCR and quantitative real-time PCR is given in: Freeman, W. M. et al., 1999, Biotechniques, 26, 112-125 and Bustin, S. A., 2000, J. Mol. Endocrinol., 25, 169-193). Most methods rely on the use of an oligonucleotide labelled with a fluorophore and a quencher moiety. The quencher reduces the fluorescence of the fluorophore by fluorescence resonance energy transfer (FRET) when the two moieties are separated by <100 Å (Clegg, R. M., 1992, Methods Enzymol., 211, 353-388). During PCR, the fluorophore and quencher are separated in space causing an increase in fluorescence. The separation occurs either by cleavage of the oligonucleotide also referred to as 5′-3′ exonuclease assay, or by a change in secondary structure of the oligonucleotide probe when it anneals to target DNA, as occurs with molecular beacons or scorpion primers (For a recent, although not complete, review on recent probe technologies see Didenko, V. D., 2001, Biotechniques, 31: 1106-1121). Quantitative, real-time, multiplex PCR with two sets of differently labeled gene-specific probes is useful because one primer/probe set may be used to detect the amount of a gene that is variable and another to detect a gene that is relatively constant and is used as a reference for the quantification of the variable gene. When both, the low and high copy target nucleic acids are amplified in one reaction, amplification of the low copy target nucleic acid is often interfered by the amplification of the high copy target gene due to the saturation of the amplifying enzyme (such as DNA polymerase) by the high copy target nucleic acid during the later cycles of amplification. Therefore, failure of quantification or inaccurate quantification is often the result of such assays making comparison between different nucleic acid samples difficult. This can dramatically affect the interpretation of experimental or clinical results such as the wrong estimation of the viral load in the blood of a patient undergoing therapy.
However, co-amplification reactions do not only possess a problem for reactions in which the nucleic acids to be amplified exist at varying copy numbers such as in quantitative PCR or RT-PCR. Simultaneous amplification of nucleic acid molecules which are contained in a nucleic acid sample is often compromised by competition of the various amplification products for limiting reaction parameters. Such applications comprise e.g. the co-amplification of genetic markers for determining genomic variabilities such as short tandem repeats (STRs), variable number tandem repeats (VNTRs) or single nucleotide polymorphisms (SNPs). The determination of these genomic variabilities are increasingly used in research and diagnostic applications such as drug discovery, pharmacology, patient management, population genetics, genotyping e.g. for paternity testing, forensics, pathology and breeding analysis.
Various solutions to this problem have been proposed for PCR, including adjusting the concentrations of the primers, use of different polymerase amounts, specialised reaction buffer formulations or utilizing primer sets with specific melting temperatures (Tm's), or combinations thereof. Adjusting the primer ratios has been referred in the art as “primer biasing” the PCR yield, and requires a decrease in the concentration of primers for the high copy target nucleic acid or of the nucleic acid molecule which is preferentially amplified. Only modest control of the process is achieved with this approach.
Another approach to co-amplification has been to adjust the temperature of annealing in PCR such that the primers for the high copy target nucleic acid or the preferentially amplified nucleic acid anneal to a lesser extent than those for the low copy target nucleic acid or the inefficient amplified nucleic acid. This approach also has a problem. The Tm difference between primer pairs must be relatively large before good modulation of PCR can be exerted on the differential yields for the high and low copy nucleic acids. Exact Tm's cannot be calculated (although they can be estimated), and thus they must be measured. This requires a high degree of effort, and are considerably tedious.
Alternatively, adding time to the priming or extension steps in PCR in all cycles can minimize the DNA polymerase saturation by the high copy target nucleic acid or the more efficiently amplified nucleic acid and increase amplification efficiency. However, this solution has limited utility in situations where many nucleic acids which are present in varying concentrations, are being amplified simultaneously or if amplification products differ significantly in size.
It is known that the hybridization rate of nucleic acids is increased considerably in the presence of volume exclusion agents such as dextran sulfate or polyethylene glycol due to exclusion of nucleic acids from the volume of solution occupied by the agents “Sambrook et al, Molecular Cloning, A Laboratory Manual”, page 9.50, 1989, and U.S. Pat. No. 5,106,730 (Van Ness et al.). This exclusion effect increases the effective concentration of the nucleic acids in the solution, thereby increasing the rate of hybridization. Thus, such materials are routinely added to reaction mixtures to “drive” unfavourable reactions forward. For example, they are added to reaction mixtures to “drive” ligase reactions, U.S. Pat. No. 5,185,243 (Ullman et al.) and U.S. Pat. No. 5,194,370 (Berninger et al.).
All patents, patent applications, and publications mentioned herein, both supra and infra, are incorporated herein by reference.